A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Evidence for Mucin-Like Glycoproteins That Tether Sporozoites of Cryptosporidium parvum to the Inner Surface of the Oocyst Wall

Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls.Cryptosporidium parvum and the related species Cryptosporidium hominis are apicomplexan parasites, which are spread by the fecal-oral route in contaminated water and cause diarrhea, particularly in immunocompromised hosts (1, 12, 39, 47). The infectious and diagnostic form of C. parvum is the oocyst, which has a single, multilayered, spherical wall that surrounds four sporozoites, the invasive forms (14, 27, 31). The outermost layer of the C. parvum oocyst wall is most often absent from electron micrographs, as it is labile to bleach used to remove contaminating bacteria from C. parvum oocysts (27). We will refer to this layer as the outer veil, which is the term used for a structure with an identical appearance on the surface of the oocyst wall of another apicomplexan parasite, Toxoplasma gondii (10). At the center of the C. parvum oocyst wall is a protease-resistant and rigid bilayer that contains GalNAc (5, 23, 43). When excysting sporozoites break through the oocyst wall, the broken edges of this bilayer curl in, while the overall shape of the oocyst wall remains spherical.The inner, moderately electron-dense layer of the C. parvum oocyst wall is where the Cryptosporidium oocyst wall proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) have been localized with monoclonal antibodies (4, 20, 28, 32). COWPs, which have homologues in Toxoplasma, are a family of nine proteins that contain polymorphic Cys-rich and His-rich repeats (37, 46). Finally, on the inner surface of C. parvum oocyst walls are knob-like structures, which cross-react with an anti-oocyst monoclonal antibody (11).Like other apicomplexa (e.g., Toxoplasma and Plasmodium), sporozoites of C. parvum are slender, move by gliding motility, and release adhesins from apical organelles when they invade host epithelial cells (1, 8, 12, 39). Unlike other apicomplexa, C. parvum parasites are missing a chloroplast-derived organelle called the apicoplast (1, 47, 49). C. parvum sporozoites have on their surface unique mucin-like glycoproteins, which contain Ser- and Thr-rich repeats that are polymorphic and may be modified by O-linked GalNAc (4-7, 21, 25, 26, 30, 32, 34, 35, 43, 45). These C. parvum mucins, which are highly immunogenic and are potentially important vaccine candidates, include gp900 and gp40/gp15 (4, 6, 7, 25, 26). gp40/gp15 is cleaved by furin-like proteases into two peptides (gp40 and gp15), each of which is antigenic (42). gp900, gp40, and gp15 are shed from the surface of the C. parvum sporozoites during gliding motility (4, 7, 35).The studies presented here began with electron microscopic observations of C. parvum oocysts stained with ruthenium red (23), which revealed novel fibrils or tethers that extend radially from the inner surface of the oocyst wall to the outer surface of sporozoites. We hypothesized that at least some of these fibrillar tethers might be the antigenic mucins, which are abundant on the surface of C. parvum sporozoites. To test this hypothesis, we used mass spectroscopy to identify oocyst wall proteins and sporozoite glycoproteins and used deconvolving and immunoelectron microscopy (immuno-EM) with lectins and anti-C. parvum antibodies to directly label the tethers.     

The Genome of the Amoeba Symbiont “Candidatus Amoebophilus asiaticus” Reveals Common Mechanisms for Host Cell Interaction among Amoeba-Associated Bacteria

Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of “Candidatus Amoebophilus asiaticus,” an obligate intracellular amoeba symbiont belonging to the Bacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. “Ca. Amoebophilus asiaticus” encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of “Ca. Amoebophilus asiaticus”. More generally, we show that the eukaryotic domains identified in “Ca. Amoebophilus asiaticus” are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila, Rickettsia bellii, Francisella tularensis, and Mycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.Free-living amoebae, such as Acanthamoeba spp., are ubiquitous protozoa which can be found in such diverse habitats as soil, marine water, and freshwater and in many engineered environments (62, 100). They are important predators of prokaryotic and eukaryotic microorganisms, thereby having great influence on microbial community composition, soil mineralization, plant growth, and nutrient cycles (14, 100). Interestingly, many well-known pathogens of humans are able to infect, survive, and multiply within amoebae (39, 51). These protozoa can thus serve as reservoirs and vectors for the transmission of pathogenic bacteria to humans, as demonstrated for L. pneumophila and Mycobacterium avium (2, 115). It is also increasingly being recognized that protozoa are important for the adaptation of (pathogenic) bacteria to higher eukaryotic cells as a niche for growth (2, 24, 42, 78, 89).In addition to the many recognized transient associations between amoeba and pathogens, stable and obligate relationships between bacteria and amoebae also were described for members of the Alphaproteobacteria (11, 34, 48), the Betaproteobacteria (49), the Bacteroidetes (50), and the Chlamydiae (4, 12, 35, 52). These obligate amoeba symbionts show a worldwide distribution, since phylogenetically highly similar strains were found in amoeba isolates from geographically distant sources (51, 107). The phylogenetic diversity and the different lifestyles of these obligate intracellular bacteria—some are located directly in the host cell cytoplasm (11, 34, 48-50, 52), while others are enclosed in host-derived vacuoles (4, 35, 44)—suggest fundamentally different mechanisms of host cell interaction. However, with the exception of chlamydia-related amoeba symbionts (37, 46, 47), our knowledge of the biology of obligate intracellular symbionts of amoebae is still scarce.Comparative genomics has been extremely helpful for the analysis of intracellular bacteria. Numerous genome sequences from the Alpha- and Gammaproteobacteria and the Chlamydiae are available and have contributed significantly to our understanding of genome evolution, the biology of intracellular bacteria, and the interactions with their host cells (24, 26, 46, 79, 82). In this study, we determined and analyzed the complete genome sequence of “Candidatus Amoebophilus asiaticus” strain 5a2 in order to gain novel insights into its biology. “Ca. Amoebophilus asiaticus” is a Gram-negative, obligate intracellular amoeba symbiont belonging to the Bacteroidetes which has been discovered within an amoeba isolated from lake sediment (107). “Ca. Amoebophilus asiaticus” shows highest 16S rRNA similarity to “Candidatus Cardinium hertigii,” an obligate intracellular parasite of arthropods able to manipulate the reproduction of its hosts (131). According to 16S rRNA trees, both organisms are members of a monophyletic group within the phylogenetically diverse phylum Bacteroidetes, consisting only of symbionts and sequences which were directly retrieved from corals (113). Among members of the Bacteroidetes, the genome sequences of only three symbionts, which are only distantly related (75 to 80% 16S rRNA sequence similarity) to “Ca. Amoebophilus asiaticus,” have been determined to date: two strains of “Candidatus Sulcia muelleri,” a symbiont of sharpshooters, and “Azobacteroides pseudotrichonymphae,” a symbiont of an anaerobic termite gut ciliate (45, 72, 74, 127).The genome of “Ca. Amoebophilus asiaticus” is only moderately reduced in size compared to those of many other obligate intracellular bacteria (75, 123), but nevertheless, its biosynthetic capabilities are extremely limited. A large fraction of the genome consists of IS elements and an unparalleled high number of proteins with eukaryotic domains, such as ankyrin repeats, TPR/SEL1 repeats, leucine-rich repeats, and domains from the eukaryotic ubiquitin system, all of them most likely important for host cell interaction. Feature enrichment analysis across a nonredundant data set of all bacterial genomes showed that these domains are enriched in the genomes of bacteria (including several pathogens of humans) known to be able to infect amoebae, providing further evidence for an important role of amoebae in the evolution of mechanisms for host cell interaction in intracellular bacteria.     

Cre-lox-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of Magnetospirillum gryphiswaldense

Magnetosome biomineralization and magnetotaxis in magnetotactic bacteria are controlled by numerous, mostly unknown gene functions that are predominantly encoded by several operons located within the genomic magnetosome island (MAI). Genetic analysis of magnetotactic bacteria has remained difficult and requires the development of novel tools. We established a Cre-lox-based deletion method which allows the excision of large genomic fragments in Magnetospirillum gryphiswaldense. Two conjugative suicide plasmids harboring lox sites that flanked the target region were subsequently inserted into the chromosome by homologous recombination, requiring only one single-crossover event, respectively, and resulting in a double cointegrate. Excision of the targeted chromosomal segment that included the inserted plasmids and their resistance markers was induced by trans expression of Cre recombinase, which leaves behind a scar of only a single loxP site. The Cre helper plasmid was then cured from the deletant strain by relief of antibiotic selection. We have used this method for the deletion of 16.3-kb, 61-kb, and 67.3-kb fragments from the genomic MAI, either in a single round or in subsequent rounds of deletion, covering a region of approximately 87 kb that comprises the mamAB, mms6, and mamGFDC operons. As expected, all mutants were Mag⁻ and some were Mot⁻; otherwise, they showed normal growth patterns, which indicates that the deleted region is not essential for viability in the laboratory. The method will facilitate future functional analysis of magnetosome genes and also can be utilized for large-scale genome engineering in magnetotactic bacteria.Magnetosomes are unique membrane-enveloped organelles that are formed by magnetotactic bacteria (MTB) for magnetic navigation (2, 37). The mechanism of magnetosome formation is within the focus of a multidisciplinary interest and has relevance for biotechnological applications (5). It has been recognized that the biomineralization of inorganic magnetite crystals and their assembly into highly ordered magnetosome chains are under strict genetic control. Recent studies combining proteomic and bioinformatic approaches suggested that the genetic determination of magnetosome formation is complex and may potentially involve 25 to 50 gene functions (15), with unknown numbers of accessory genes and those controlling signal transduction and motility to achieve effective magnetotaxis (8, 9, 12, 26, 27, 29). However, the functional characterization of these candidate genes has been lagging behind. This is due to technical difficulties and the lack of facile tools for genetic manipulation of MTB. Allelic replacement systems have been established for Magnetospirillum magneticum (18) and Magnetospirillum gryphiswaldense (39, 40), but so far, there are only few examples of these for magnetosome genes that were functionally characterized because of the tedious and cumbersome procedures required for mutant generation (11, 19, 28, 31-32). Most genes controlling magnetosome formation in these and other MTB are located within a genomic magnetosome island (MAI) (34), which is genetically instable during stationary growth (47) and more or less conserved in other MTB (12, 13, 35). Most known magnetosome genes are organized within several conserved operons, which are interspersed with large, poorly conserved genome sections of unknown functions that have been speculated to represent genetic junk irrelevant for magnetotaxis but to cause genetic instability by their high content of repeats and transposable elements (34, 47). Thus, for large-scale functional genome analysis and rearrangements of the MAI, there is a great need for additional and more efficient genetic methods.Artificial genome recombination systems have been described for a number of bacteria. Many of them are based on the Cre-loxP system of the P1 phage (42). The Cre-loxP recombination system is a simple two-component system that is recognized as a powerful genetic tool in a multitude of eukaryotic and prokaryotic organisms (4, 6, 48). The Cre protein belongs to the integrase family of site-specific recombinases and catalyzes reciprocal site-specific recombination of DNA at 34-bp loxP sites, resulting in either excision or inversion, depending on the parallel or antiparallel orientation of the loxP sites, respectively (21). It does not require any host cofactors or accessory proteins (7). Cre-lox deletion has several advantages over other methods, such as a high efficiency and the independency of the length of DNA located between the two lox sites. The utility of Cre-lox systems has been demonstrated in a wide variety of Gram-positive and Gram-negative bacteria (17, 22-23). In several studies, it was applied for the generation of large-scale deletions, as in for example, the Gram-positive Corynebacterium glutamicum (43-46) and Bacillus subtilis (49).In M. gryphiswaldense, the functionality of a Cre-loxP antibiotic marker recycling system (25) has been previously demonstrated by deletion of a single gene based on double-crossover insertion of two loxP sites, followed by subsequent Cre-mediated excision (31). In this study, we describe a novel strategy for Cre-loxP-mediated deletion of large genomic fragments which requires only two single crossovers. The system has been validated by the generation of three large deletions, two single and one combination within the MAI, which demonstrated that the total deleted region of approximately 87 kb is not essential for viability and growth in the laboratory.     

Optical Mapping Reveals a Large Genetic Inversion between Two Methicillin-Resistant Staphylococcus aureus Strains

Staphylococcus aureus is a highly versatile and evolving bacterium of great clinical importance. S. aureus can evolve by acquiring single nucleotide polymorphisms and mobile genetic elements and by recombination events. Identification and location of novel genomic elements in a bacterial genome are not straightforward, unless the whole genome is sequenced. Optical mapping is a new tool that creates a high-resolution, in situ ordered restriction map of a bacterial genome. These maps can be used to determine genomic organization and perform comparative genomics to identify genomic rearrangements, such as insertions, deletions, duplications, and inversions, compared to an in silico (virtual) restriction map of a known genome sequence. Using this technology, we report here the identification, approximate location, and characterization of a genetic inversion of ∼500 kb of a DNA element between the NRS387 (USA800) and FPR3757 (USA300) strains. The presence of the inversion and location of its junction sites were confirmed by site-specific PCR and sequencing. At both the left and right junction sites in NRS387, an IS1181 element and a 73-bp sequence were identified as inverted repeats, which could explain the possible mechanism of the inversion event.Staphylococcus aureus is a gram-positive bacterium of immense clinical importance. This opportunistic pathogen is capable of causing a wide range of diseases from skin and soft-tissue infections to life-threatening bacteremia, endocarditis, and osteomyelitis (14). Approximately 75% of the S. aureus genome is composed of a core genome that is common in all strains, and 25% of the genome is composed of variable regions which can differ between different strains (4, 16, 24-26). S. aureus evolves primarily by introducing single nucleotide polymorphisms in its core genome and by acquiring mobile genetic elements (MGEs) through horizontal gene transfer. These MGEs include pathogenicity/genomic islands, plasmids, transposons, and bacteriophages that become integrated in the chromosome (4, 11, 16, 31, 32). Despite being a heterogeneous organism, genetic recombination in S. aureus is proposed to be rather rare (20, 24, 29, 35). Its clones are more likely to evolve by point mutations than by recombination events (12). The MGEs contribute to the phenotypic and genotypic diversity seen with the S. aureus population. Acquisition of the staphylococcal cassette chromosome (SCCmec) elements through site-specific recombinases has led to the epidemic of methicillin-resistant S. aureus (MRSA) strains in hospitals and communities all over the world (6, 10, 15). In recent years, the integration of arginine catabolite mobile element in the USA300 lineage of MRSA has been proposed to give the pathogen its epidemiological advantage, including traits for surviving in low-pH conditions and oxygen tension environments (11). In addition, chromosomal replacements have been observed within lineages of sequence type 34 (ST34) and ST42 (34) and ST8 and ST30 (13).Genomic rearrangements, such as inversions, have been observed with genomes of enteric bacteria, such as Salmonella enterica, Shigella flexneri, Yersinia pestis KIM, Escherichia coli (K12 and O157:H7), and group A Streptococcus pyogenes (8, 9, 18, 27, 28, 30, 37). No genomic inversions in S. aureus have been reported to date. With the use of optical mapping, large genomic rearrangements, such as inversions, that would otherwise be missed with other comparative genotyping approaches, including microarray analysis, can be identified. Optical mapping uses high-resolution restriction maps (optical maps) of a bacterial genome to determine its genomic organization (5, 21, 23, 33, 36). These optical maps can be compared to an in silico (virtual) restriction map of a known genome sequence and can be used to identify gene rearrangements and their locations. Using optical mapping in conjunction with subsequent site-specific PCR and sequencing, we report the identification, approximate location, and partial characterization of an ∼500-kb DNA element in NRS387, a USA800 strain that was found to be inverted relative to USA300FPR3757. Identification of IS1181 elements and a novel 73-bp element at both ends of the ∼500-kb element in NRS387 could suggest the possibility of an inversion event in an ancestral strain of NRS387.     

Spontaneous Excision of the Salmonella enterica Serovar Enteritidis-Specific Defective Prophage-Like Element φSE14

Salmonella enterica serovar Enteritidis has emerged as a major health problem worldwide in the last few decades. DNA loci unique to S. Enteritidis can provide markers for detection of this pathogen and may reveal pathogenic mechanisms restricted to this serovar. An in silico comparison of 16 Salmonella genomic sequences revealed the presence of an ∼12.5-kb genomic island (GEI) specific to the sequenced S. Enteritidis strain NCTC13349. The GEI is inserted at the 5′ end of gene ydaO (SEN1377), is flanked by 308-bp imperfect direct repeats (attL and attR), and includes 21 open reading frames (SEN1378 to SEN1398), encoding primarily phage-related proteins. Accordingly, this GEI has been annotated as the defective prophage SE14 in the genome of strain NCTC13349. The genetic structure and location of φSE14 are conserved in 99 of 103 wild-type strains of S. Enteritidis studied here, including reference strains NCTC13349 and LK5. Notably, an extrachromosomal circular form of φSE14 was detected in every strain carrying this island. The presence of attP sites in the circular forms detected in NCTC13349 and LK5 was confirmed. In addition, we observed spontaneous loss of a tetRA-tagged version of φSE14, leaving an empty attB site in the genome of strain NCTC13349. Collectively, these results demonstrate that φSE14 is an unstable genetic element that undergoes spontaneous excision under standard growth conditions. An internal fragment of φSE14 designated Sdf I has been used as a serovar-specific genetic marker in PCR-based detection systems and as a tool to determine S. Enteritidis levels in experimental infections. The instability of this region may require a reassessment of its suitability for such applications.The genus Salmonella comprises a heterogeneous group of Gram-negative bacteria, differentiable by biochemical and serological properties. More than 2,500 Salmonella serovars have been identified according to the serospecificities of the somatic and flagellar antigens. Some serovars, exemplified by Salmonella enterica serovar Typhimurium and S. Enteritidis, can infect a broad range of hosts. However, a subset of serovars, such as S. Typhi, a human-specific pathogen, show a high degree of adaptation to a specific host.In the last few decades, S. Enteritidis has emerged as a major health problem worldwide (31). This pathogen colonizes the reproductive organs of infected birds without causing discernible illness and survives host defenses during the formation of the egg (25, 27). The production of a capsule-like O antigen structure by certain wild-type strains of S. Enteritidis (30, 46) has been associated with reproductive tract tropism and improved survival within eggs (26, 27, 45). Egg contamination can originate before oviposition by direct contamination of the yolk, albumen, or eggshell membranes with bacteria from the infected reproductive organs of the birds or after or during oviposition by penetration of bacteria from contaminated feces through the eggshell (8, 14, 25). Transmission of the bacterium to humans occurs mainly through the consumption of contaminated eggs or egg products (8, 14, 25). Upon infection of a human host, S. Enteritidis causes self-limiting gastroenteritis similar to that caused by other nontyphoidal Salmonella serovars.According to information gathered from 84 countries responding to a global survey conducted by the World Health Organization (WHO), S. Enteritidis and S. Typhimurium accounted for ∼70% of all human and nonhuman isolates of Salmonella reported worldwide between 1995 and 2008. In fact, S. Enteritidis alone accounted for 61.4% of the ∼1.5 million human isolates of Salmonella reported during this period, according to the WHO Global Foodborne Infections Network Country Databank (http://www.who.int/salmsurv). Remarkably, S. Enteritidis is the second most prevalent cause of Salmonella infection in humans, after S. Typhimurium, in the United States (10).The high global prevalence of S. Enteritidis makes the development of a rapid, sensitive, and highly specific detection system critical to collect accurate epidemiologic data. The identification of loci that serve as specific markers for DNA-based identification of this pathogen may also provide insights into pathogenic mechanisms restricted to this serovar. Genomic regions that are unique to given serovars are especially suitable for such epidemiologic detection (3). For instance, Agron and colleagues identified an S. Enteritidis-specific genomic region of ∼4,060 bp adjacent to the ydaO gene, carrying six open reading frames (ORFs) that they designated lygA to lygF (1). A PCR-based assay successfully detected the presence of an internal fragment of this serovar-specific region in most strains in a diverse collection of clinical and environmental S. Enteritidis isolates and not in 73 non-Enteritidis isolates of S. enterica representing 34 different serovars (1). Since then, this region has been widely used as an S. Enteritidis-specific molecular marker in the development of several PCR-based assays for detection and epidemiological typing of Salmonella serovars in clinical and environmental samples (2, 11, 32, 37, 44, 53). Recently, an S. Enteritidis-specific real-time quantitative PCR (qPCR) assay based on the detection of this region was developed (15). This qPCR assay has been used in a series of studies of the distribution and replication kinetics of S. Enteritidis in experimentally infected animals (16-21).We performed a bioinformatic study to identify genomic regions specific to S. Enteritidis and found a genomic island (GEI) that includes the S. Enteritidis-specific locus lyg (1). This island has been annotated as the defective prophage SE14 in the genome of S. Enteritidis strain NCTC13349 (52). Although we demonstrate that the location in the genome and the overall genetic structure of the island are conserved in wild-type isolates of S. Enteritidis from different origins, we detected strains that do not carry the island in their genomes. Finally, we demonstrate here that the island corresponds to an unstable element that undergoes spontaneous excision from the genome of S. Enteritidis under standard growth conditions.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

Bocavirus Infection Induces Mitochondrion-Mediated Apoptosis and Cell Cycle Arrest at G2/M Phase

Bocavirus is a newly classified genus of the family Parvovirinae. Infection with Bocavirus minute virus of canines (MVC) produces a strong cytopathic effect in permissive Walter Reed/3873D (WRD) canine cells. We have systematically characterized the MVC infection-produced cytopathic effect in WRD cells, namely, the cell death and cell cycle arrest, and carefully examined how MVC infection induces the cytopathic effect. We found that MVC infection induces an apoptotic cell death characterized by Bax translocalization to the mitochondrial outer membrane, disruption of the mitochondrial outer membrane potential, and caspase activation. Moreover, we observed that the activation of caspases occurred only when the MVC genome was replicating, suggesting that replication of the MVC genome induces apoptosis. MVC infection also induced a gradual cell cycle arrest from the S phase in early infection to the G₂/M phase at a later stage, which was confirmed by the upregulation of cyclin B1 and phosphorylation of cdc2. Cell cycle arrest at the G₂/M phase was reproduced by transfection of a nonreplicative NS1 knockout mutant of the MVC infectious clone, as well as by inoculation of UV-irradiated MVC. In contrast with other parvoviruses, only expression of the MVC proteins by transfection did not induce apoptosis or cell cycle arrest. Taken together, our results demonstrate that MVC infection induces a mitochondrion-mediated apoptosis that is dependent on the replication of the viral genome, and the MVC genome per se is able to arrest the cell cycle at the G₂/M phase. Our results may shed light on the molecular pathogenesis of Bocavirus infection in general.The Bocavirus genus is newly classified within the subfamily Parvovirinae of the family Parvoviridae (21). The currently known members of the Bocavirus genus include bovine parvovirus type 1 (BPV1) (17), minute virus of canines (MVC) (57), and the recently identified human bocaviruses (HBoV, HBoV2, and HBoV3) (4, 7, 36).MVC was first recovered from canine fecal samples in 1970 (10). The virus causes respiratory disease with breathing difficulty (14, 32, 49) and enteritis with severe diarrhea (11, 39), which often occurs with coinfection with other viruses (39), spontaneous abortion of fetuses, and death of newborn puppies (14, 29). Pathological lesions in fetuses in experimental infections were found in the lymphoid tissue of the lung and small intestine (14). MVC was isolated and grown in the Walter Reed/3873D (WRD) canine cell line (10), which is derived from a subdermoid cyst of an irradiated male dog (10). The full-length 5.4-kb genome of MVC was recently mapped with palindromic termini (60). Under the control of a single P6 promoter, through the mechanism of alternative splicing and alternative polyadenylation, MVC expresses two nonstructural proteins (NS1 and NP1) and two capsid proteins (VP1 and VP2). Like the NS1 proteins of other parvoviruses, the NS1 of MVC is indispensable for genome replication. The NP1 protein, which is unique to the Bocavirus genus, appears to be critical for optimal viral replication, as the NP1 knockout mutant of MVC suffers from severe impairment of replication (60). A severe cytopathic effect during MVC infection of WRD cells has been documented (10, 60).The HBoV genome has been frequently detected worldwide in respiratory specimens from children under 2 years old with acute respiratory illnesses (2, 34, 55). HBoV is associated with acute expiratory wheezing and pneumonia (3, 34, 55) and is commonly detected in association with other respiratory viruses (34, 55). Further studies are necessary, however, to identify potential associations of HBoV infection with clinical symptoms or disease of acute gastroenteritis (7, 36). The full-length sequence of infectious MVC DNA (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ214110","term_id":"219665308","term_text":"FJ214110"}}FJ214110) that we have reported shows 52.6% identity to HBoV, while the NS1, NP1, and VP1 proteins are 38.5%, 39.9%, and 43.7% identical to those of HBoV, respectively (60).The cytopathic effect induced during parvovirus infection has been widely documented, e.g., in infections with minute virus of mice (MVM) (13), human parvovirus B19 (B19V) (58), parvovirus H-1 (25, 52), and BPV1 (1). In Bocavirus, cell death during BPV1 infection of embryonic bovine tracheal cells has been shown to be achieved through necrosis, independent of apoptosis (1). B19V-induced cell death of primary erythroid progenitor cells has been shown to be mainly mediated by an apoptotic pathway (58) in which the nonstructural protein 11kDa plays a key role (16). In contrast, the MVM-induced cytopathic effect has been revealed to be mediated by NS1 interference with intracellular casein kinase II (CKII) signaling (22, 44, 45), a nonapoptotic cell death. Oncolytic parvovirus H-1 infections can induce either apoptosis or nonapoptotic cell death, depending on the cell type (25, 40). Therefore, the mechanisms underlying parvovirus infection-induced cell death vary, although NS1 has been widely shown to be involved in both apoptotic and nonapoptotic cell death. The nature of the cytopathic effect during Bocavirus MVC infection has not been studied.Parvovirus replication requires infected cells at the S phase. Infection with parvovirus has been revealed to accompany a cell cycle perturbation that mostly leads to an arrest in the S/G₂ phase or the G₂/M phase during infection (30, 33, 42, 47, 65). MVM NS1 expression induces an accumulation of sensitive cells in the S/G₂ phase (6, 46, 47). Whether MVC infection-induced cell death is accompanied by an alternation of cell cycle progression and whether the viral nonstructural protein is involved in these processes have not been addressed.In this study, we found, in contrast with other members of the family Parvoviridae, expression of both the nonstructural and structural proteins of MVC by transfection did not induce cell death or cell cycle arrest. However, the cytopathic effect induced during MVC infection is a replication-coupled, mitochondrion-mediated and caspase-dependent apoptosis, accompanied with a gradual cell cycle arrest from the S phase to the G₂/M phase, which is facilitated by the MVC genome.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

An IcmF Family Protein,ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL,and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL_M, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL_M and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL_M is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.Many pathogenic gram-negative bacteria employ protein secretion systems formed by macromolecular complexes to deliver proteins or protein-DNA complexes across the bacterial membrane. In addition to the general secretory (Sec) pathway (18, 52) and twin-arginine translocation (Tat) pathway (7, 34), which transport proteins across the inner membrane into the periplasm, at least six distinct protein secretion systems occur in gram-negative bacteria (28, 46, 66). These systems are able to secrete proteins from the cytoplasm or periplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (10, 15, 23, 26, 30) and a recently discovered type VI secretion system (T6SS) (4, 8, 22, 41, 48, 49). These systems use ATPase or a proton motive force to energize assembly of the protein secretion machinery and/or substrate translocation (2, 6, 41, 44, 60).Agrobacterium tumefaciens is a soilborne pathogenic gram-negative bacterium that causes crown gall disease in a wide range of plants. Using an archetypal T4SS (9), A. tumefaciens translocates oncogenic transferred DNA and effector proteins to the host and ultimately integrates transferred DNA into the host genome. Because of its unique interkingdom DNA transfer, this bacterium has been extensively studied and used to transform foreign DNA into plants and fungi (11, 24, 40, 67). In addition to the T4SS, A. tumefaciens encodes several other secretion systems, including the Sec pathway, the Tat pathway, T1SS, T5SS, and the recently identified T6SS (72). T6SS is highly conserved and widely distributed in animal- and plant-associated Proteobacteria and plays an important role in the virulence of several human and animal pathogens (14, 19, 41, 48, 56, 63, 74). However, T6SS seems to play only a minor role or even a negative role in infection or virulence of the plant-associated pathogens or symbionts studied to date (5, 37-39, 72).T6SS was initially designated IAHP (IcmF-associated homologous protein) clusters (13). Before T6SS was documented by Pukatzki et al. in Vibrio cholerae (48), mutations in this gene cluster in the plant symbiont Rhizobium leguminosarum (5) and the fish pathogen Edwardsiella tarda (51) caused defects in protein secretion. In V. cholerae, T6SS was responsible for the loss of cytotoxicity for amoebae and for secretion of two proteins lacking a signal peptide, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein (VgrG). Secretion of Hcp is the hallmark of T6SS. Interestingly, mutation of hcp blocks the secretion of VgrG proteins (VgrG-1, VgrG-2, and VgrG-3), and, conversely, vgrG-1 and vgrG-2 are both required for secretion of the Hcp and VgrG proteins from V. cholerae (47, 48). Similarly, a requirement of Hcp for VgrG secretion and a requirement of VgrG for Hcp secretion have also been shown for E. tarda (74). Because Hcp forms a hexameric ring (41) stacked in a tube-like structure in vitro (3, 35) and VgrG has a predicted trimeric phage tail spike-like structure similar to that of the T4 phage gp5-gp27 complex (47), Hcp and VgrG have been postulated to form an extracellular translocon. This model is further supported by two recent crystallography studies showing that Hcp, VgrG, and a T4 phage gp25-like protein resembled membrane penetration tails of bacteriophages (35, 45).Little is known about the topology and structure of T6SS machinery subunits and the distinction between genes encoding machinery subunits and genes encoding regulatory proteins. Posttranslational regulation via the phosphorylation of Fha1 by a serine-threonine kinase (PpkA) is required for Hcp secretion from Pseudomonas aeruginosa (42). Genetic evidence for P. aeruginosa suggested that the T6SS may utilize a ClpV-like AAA⁺ ATPase to provide the energy for machinery assembly or substrate translocation (41). A recent study of V. cholerae suggested that ClpV ATPase activity is responsible for remodeling the VipA/VipB tubules which are crucial for type VI substrate secretion (6). An outer membrane lipoprotein, SciN, is an essential T6SS component for mediating Hcp secretion from enteroaggregative Escherichia coli (1). A systematic study of the T6SS machinery in E. tarda revealed that 13 of 16 genes in the evp gene cluster are essential for secretion of T6S substrates (74), which suggests the core components of the T6SS. Interestingly, most of the core components conserved in T6SS are predicted soluble proteins without recognizable signal peptide and transmembrane (TM) domains.The intracellular multiplication F (IcmF) and H (IcmH) proteins are among the few core components with obvious TM domains (8). In Legionella pneumophila Dot/Icm T4SSb, IcmF and IcmH are both membrane localized and partially required for L. pneumophila replication in macrophages (58, 70, 75). IcmF and IcmH are thought to interact with each other in stabilizing the T4SS complex in L. pneumophila (58). In T6SS, IcmF is one of the essential components required for secretion of Hcp from several animal pathogens, including V. cholerae (48), Aeromonas hydrophila (63), E. tarda (74), and P. aeruginosa (41), as well as the plant pathogens A. tumefaciens (72) and Pectobacterium atrosepticum (39). In E. tarda, IcmF (EvpO) interacted with IcmH (EvpN), EvpL, and EvpA in a yeast two-hybrid assay, and its putative nucleotide-binding site (Walker A motif) was not essential for secretion of T6SS substrates (74).In this study, we characterized the topology and interactions of the IcmF and IcmH family proteins ImpL_M and ImpK_L, which are two essential components of the T6SS of A. tumefaciens. We adapted the nomenclature proposed by Cascales (8), using the annotated gene designation followed by the letter indicated by Shalom et al. (59). Our data indicate that ImpL_M and ImpK_L are both integral inner membrane proteins and interact with each other via their N-terminal domains residing in the cytoplasm. We also provide genetic evidence showing that ImpL_M may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion.